Presentation on theme: "Fluorescence, Phosphorescence, & Chemiluminescence"— Presentation transcript:
1 Fluorescence, Phosphorescence, & Chemiluminescence A) Introduction1.) Theory of Fluorescence and Phosphorescence:10-5 to 10-8 s fluorescence10-4 to 10s phosphorescence10-14 to s10-8 – 10-9sM* M + heat- Excitation of e- by absorbance of hn.- Re-emission of hn as e- goes to ground state.Use hn2 for qualitative and quantitative analysis
2 Fluorescence, Phosphorescence, & Chemiluminescence A) Introduction1.) Theory of Fluorescence and Phosphorescence:MethodMass detection limit (moles)Concentration detection limit (molar)AdvantagesUV-Vis10-13 to 10-1610-5 to 10-8Universalfluorescence10-15 to 10-1710-7 to 10-9SensitiveFor UV/Vis need to observe Po and P difference, which limits detectionFor fluorescence, only observe amount of PL
3 Example of Phosphorescence 2.) Fluorescence – ground state to single state and back.Phosphorescence - ground state to triplet state and back.10-5 to 10-8 s10-4 to 10 sSpins pairedNo net magnetic fieldSpins unpairednet magnetic fieldFluorescencePhosphorescence0 sec1 sec640 secExample of Phosphorescence
4 3) Jablonski Energy Diagram S2, S1 = Singlet StatesT1 = Triplet StateNumerous vibrational energy levels for each electronic stateResonance Radiation - reemission at same lusually reemission at higher l (lower energy)Forbidden transition: no direct excitation of triplet state because change in multiplicity –selection rules.
5 4.) Deactivation Processes: a) vibrational relaxation: solvent collisions- vibrational relaxation is efficient and goes to lowest vibrational level of electronic state within 10-12s or less.- significantly shorter life-time then electronically excited state- fluorescence occurs from lowest vibrational level of electronic excited state, but can go to higher vibrational state of ground level.- dissociation: excitation to vibrational state with enough energy to break a bond- predissociation: relaxation to vibrational state with enough energy to break a bond
6 4.) Deactivation Processes: b) internal conversion: not well understood- crossing of e- to lower electronic state.- efficient since many compounds don’t fluoresce- especially probable if vibrational levels of two electronic states overlap, can lead to predissociation or dissociation.
7 4.) Deactivation Processes: c) external conversion: deactivation via collision with solvent (collisional quenching)- decrease collision increase fluorescence or phosphorescence‚ decrease temperature and/or increase viscosity‚ decrease concentration of quenching (Q) agent.Quenching of Ru(II) Luminescence by O2
8 4.) Deactivation Processes: d) intersystem crossing: spin of electron is reversed- change in multiplicity in molecule occurs (singlet to triplet)- enhanced if vibrational levels overlap- more common if molecule contains heavy atoms (I, Br)- more common in presence of paramagnetic species (O2)
9 kf + ki + kec+ kic + kpd + kd 5.) Quantum Yield (f): ratio of the number of molecules that luminesce to the total number of excited molecules.- determined by the relative rate constants (kx) of deactivation processesf = kfkf + ki + kec+ kic + kpd + kdf: fluorescence I: intersystem crossingec: external conversion ic: internal conversionpd: predissociation d: dissociationIncrease quantum yield by decreasing factors that promote other processesFluorescence probes measuring quantity of protein in a cell
10 6.) Types of Transitions: - seldom occurs from absorbance lessthan 250 nm‚ 200 nm => 600 kJ/mol, breaks many bonds- fluorescence not seen with s* s- typically p* p or p* n
11 ‚ fluorescence especially favored for rigid structures 7.) Fluorescence & Structure:- usually aromatic compounds‚ low energy of p p* transition‚ quantum yield increases with number of rings and degree of condensation.‚ fluorescence especially favored for rigid structures< fluorescence increase for chelating agent bound to metal.Examples of fluorescent compounds:quinoline indole fluorene 8-hydroxyquinoline
12 resonance forms of aniline 8.) Temperature, Solvent & pH Effects:- decrease temperature increase fluorescence- increase viscosity increase fluorescence- fluorescence is pH dependent for compounds with acidic/basic substituents.‚ more resonance forms stabilize excited state.Fluorescence pH Titrationresonance forms of aniline
13 Change in fluorescence as a function of cellular oxygen 9.) Effect of Dissolved O2:- increase [O2] decrease fluorescence‚ oxidize compound‚ paramagnetic property increase intersystem crossing (spin flipping)Change in fluorescence as a function of cellular oxygenAm J Physiol Cell Physiol 291: C781–C787, 2006.
14 B) Effect of Concentration on Fluorescence or Phosphorescence power of fluorescence emission: (F) = K’Po(1 – 10 –ebc)K’ ~ f (quantum yield)Po: power of beamebc: Beer’s lawF depends on absorbance of light and incident intensity (Po)At low concentrations: F = 2.3K’ebcPodeviations at higher concentrationscan be attributed to absorbance becominga significant factor and by self-quenchingor self-absorption.Fluorescence of crude oil
15 C) Fluorescence Spectra Excitation Spectra (a) – measure fluorescence orphosphorescence at a fixed wavelengthwhile varying the excitation wavelength.Emission Spectra (b) – measure fluorescence orphosphorescence over a range ofwavelengths using a fixed excitation wavelength.Phosphorescence bands are usually found at longer (>l) then fluorescence because excited triple state is lower energy then excited singlet state.
16 D) Instrumentation - basic design ‚ components similar to UV/Vis ‚ spectrofluorometers: observeboth excitation & emission spectra.- extra features for phosphorescence‚ sample cell in cooled Dewar flask with liquid nitrogen‚ delay between excitation and emission
18 Spectrofluorometer Perkin-Elmer 204 - both excitation and emmision spectra- two grating monochromators- quantitative analysisPerkin-Elmer 204
19 E) Application of Fluorescence - detect inorganic species by chelating ionIonReagentAbsorption (nm)Fluorescence (nm)Sensitivity (mg/ml)InterferenceAl3+Alizarin garnet R4705000.007Be, Co, Cr, Cu, F-,NO3-, Ni, PO4-3, Th, ZrF-Al complex of Alizarin garnet R (quenching)0.001Be, Co, Cr, Cu, F-,Fe, Ni,PO4-3, Th, ZrB4O72-Benzoin3704500.04Be, SbCd2+2-(0-Hydroxyphenyl)-benzoxazole365Blue2NH3Li+8-Hydroxyquinoline5800.2MgSn4+Flavanol4000.1F-, PO43-, ZrZn2+-green10B, Be, Sb, colored ions8-Hydroxyquinoline flavanol alizarin garnet R benzoin
20 F) Chemiluminescence Examples: - chemical reaction yields an electronically excited species that emitslight as it returns to ground state.- relatively new, few examplesA + B C* C + hnExamples:Chemical systems- Luminol (used to detect blood)- phenyl oxalate ester (glow sticks)
21 Luciferase gene cloned into plants 2) Biochemical systems- Luciferase (Firefly enzyme)“Glowing” PlantsLuciferase gene cloned into plantsLuciferin (firefly)